Combined toxicity of abamectin and carbendazim on enzymatic and transcriptional levels in the soil-earthworm microcosm.

To reveal the toxicological mechanisms of pesticide mixtures on soil organisms, this study concentrated on evaluating enzymatic activity and gene expression changes in the earthworm Eisenia fetida (Savigny 1826). Despite being frequently exposed to multiple pesticides, including the common combination of abamectin (ABA) and carbendazim (CAR), environmental organisms have primarily been studied for the effects of individual pesticides. Acute toxicity results exhibited that the combination of ABA and CAR caused a synergistic impact on E. fetida. The levels of MDA, ROS, T-SOD, and caspase3 demonstrated a significant increase across most individual and combined groups, indicating the induction of oxidative stress and cell death. Additionally, the expression of three genes (hsp70, gst, and crt) exhibited a significant decrease following exposure to individual pesticides and their combinations, pointing toward cellular damage and impaired detoxification function. In contrast, a noteworthy increase in ann expression was observed after exposure to both individual pesticides and their mixtures, suggesting the stimulation of reproductive capacity in E. fetida. The present findings contributed to a more comprehensive understanding of the potential toxicity mechanisms of the ABA and CAR mixture, specifically on oxidative stress, cell death, detoxification dysfunction, and reproductive capacity in earthworms. Collectively, these data offered valuable toxicological insights into the combined effects of pesticides on soil organisms, enhancing our understanding of the underlying risks associated with the coexistence of different pesticides in natural soil environments.

Characterization of the microplastic photoaging under the action of typical salt ions of biological nitrogen removal processes.

Microplastics (MPs) are one of the most prevalent and diverse contaminants, and wastewater treatment plants are significant MP aggregators. Controlling the pollution caused by microplastics requires an understanding of how they age. The properties of the MPs photoaging process under the influence of salt ions typical of biological nitrogen elimination processes were disclosed in this work. The aging process of polyvinyl chloride microplastics (PVC-MPs) was greatly slowed down by greater HCO3- and NO2- concentrations, according to a comparison of the carbonyl index changes that occurred during photoaging. The carbonyl index had a negative correlation with the thermal stability of the photo-aged PVC-MPs, and aging accelerated the elimination of chlorine from the water. The samples were aged by UV radiation after 36 h at 40 °C, and the amount of chlorine eliminated was 10.13 times greater than that of the original MPs samples. It was discovered that the leachate concentration of aged MPs dramatically increased with decreasing particle size and was positively connected with the level of aging by comparing the concentration of leachate for two particle sizes (1 mm and 100 m). Photoaging caused MPs to become rougher, which in turn improved the NO3--N, NH4+-N, and NO2--N adsorption by PVC-MPs.

YWHAH, a member of 14-3-3 family proteins, and PSME2, the proteasome activator subunit 2, are key host factors of Japanese encephalitis virus infection.

BACKGROUND: Host response to virus infection is key to the effective control and eventual elimination of viruses or infected cells; however, the underlying mechanism of Japanese encephalitis virus (JEV) infection remains unclear. METHODS: In the present study, short time-series expression was analyzed by R software to obtain two groups of differentially expressed genes (DEGs) [upregulated/downregulated] during the entire process of JEV infection based on the data in the Gene Expression Omnibus database. GO enrichment and KEGG pathway, protein interactions and hub genes selection were analyzed by DAVID, STRING and Cytoscape respectively. Interactions of the JEV and host proteins, and the microRNAs that target Tyrosine 3-monooxygenase/tryptophan 5-monooxygenase activating protein Eta (YWHAH) and Proteasome activator subunit 2(PSME2) were predicted by P-hipster and ENCORI, respectively. Expression levels of YWHAH and PSME2 were analyzed using the HPA database and RT-qPCR assay. RESULTS: Two groups of continuously changed DEGs during entire process of JEV infection were obtained. Continuously upregulated cluster was mainly related to regulation of transcription, immune response and inflammatory response; and the continuous downregulated group mainly including intracellular protein transport and signal transduction, several proteolysis pathways. As targets of several microRNAs, the downregulated-YWHAH and the upregulated-PSME2 were related to host and JEV proteins to affect several pathways after JEV infection. CONCLUSIONS: YWHAH and PSME2 are key host factors of JEV infection based on their continuously differentially expressed pattern, interactions with multiple JEV proteins, and as members of the hub genes. Our results provide valuable information for further studies on the interactions between viruses and host.

Optimal timing of treatment for errors in second language learning - A systematic review of corrective feedback timing.

Although a large body of research has developed on corrective feedback in second language acquisition (SLA) in the past 30 years, there are few empirical studies examining the relationship between feedback timing and SLA. To begin to address this gap, this study reviews the existing research on the impact of corrective feedback timing on SLA. It aims to investigate the possible influential factors that might have led to inconsistent research findings and theoretical explanations. The review was conducted according to PRISMA-statement through searches in peer-reviewed electronic databases including Social Sciences Citation Index (SSCI), Scopus, EBSCO, which includes ERIC and the British Education Index and gray literature (doctoral dissertations in ProQuest). Twenty studies conducted and published between 2006 and 2021 were finally analyzed to reveal the current trends. The results of this review indicate that there is no definite answer to the question of when errors in L2 should be treated. The difficulty of drawing a conclusive finding can be attributed to the communicative modality examined and variations in research design, including the explicitness of feedback and various ways of measuring feedback timing. No certain theoretical framework has been applied to guide these studies and they have applied different theoretical explanations to interpret the inconsistent results. The review highlights the need to continue to investigate the effectiveness of corrective feedback under different timing conditions. In addition, it discusses some research gaps that should be addressed in future studies and suggests future research directions in the area of feedback timing.

Surface figure compensation of the primary mirror in a Ritchey-Chrétien space telescope by spring preloads.

To reduce the surface figure error induced by mechanical strains during the integration process of a high-precision mirror, a cost-efficient compensation method by spring preloads is proposed. The study is based on the primary mirror of a Ritchey-Chrétien space telescope with a focal length of 1200 mm. First, the surface figure degradation of the mirror during the assembly process is expressed and analyzed. Then, a finite element model of the mirror and its mounting structure is established, and surface deformations caused by different preloading forces are simulated. An optimized combination of different preloads was obtained through data fitting, and the influence of the combined preload on the mirror was analyzed. The simulation results show that ring preloads mainly affect spherical aberration and high-order spherical aberrations, while quadrupole preloads mainly affect astigmatism, and the optimized preload can compensate for the surface figure error from 0.120λ RMS to 0.088λ RMS. Last, the surface figure error of the mirror is measured by experiments under optimized preloads, and the result is 0.084λ RMS, which verifies the correctness of the analysis process and effectiveness of the compensation method.

Diverse mechanisms associated with cyhalofop-butyl resistance in Chinese sprangletop (Leptochloa chinensis (L.) Nees): Characterization of target-site mutations and metabolic resistance-related genes in two resistant populations.

Resistance of Chinese sprangletop (Leptochloa chinensis (L.) Nees) to the herbicide cyhalofop-butyl has recently become a severe problem in rice cultivation. However, the molecular mechanisms of target-site resistance (TSR) in cyhalofop-butyl-resistant L. chinensis as well as the underlying non-target-site resistance (NTSR) have not yet been well-characterized. This study aimed to investigate cyhalofop-butyl resistance mechanisms using one susceptible population (LC-S) and two resistant populations (LC-1701 and LC-1704) of L. chinensis. We analyzed two gene copies encoding the entire carboxyltransferase (CT) domain of chloroplastic acetyl-CoA carboxylase (ACCase) from each population. Two non-synonymous substitutions were detected in the resistant L. chinensis populations (Trp2027-Cys in the ACCase1 of LC-1701 and Leu1818-Phe in the ACCase2 of LC-1704), which were absent in LC-S. As Trp2027-Cys confers resistance to ACCase-inhibiting herbicides, the potential relationship between the novel Leu1818-Phe mutation and cyhalofop-butyl resistance in LC-1704 was further explored by single-nucleotide polymorphism (SNP) detection. Metabolic inhibition assays indicated that cytochrome P450 monooxygenases (P450s) and glutathione S-transferases (GSTs) contributed to cyhalofop-butyl resistance in specific resistant populations. RNA sequencing showed that the P450 genes CYP71Z18, CYP71C4, CYP71C1, CYP81Q32, and CYP76B6 and the GST genes GSTF11, GSTF1, and GSTU6 were upregulated in at least one resistant population, which indicated their putative roles in cyhalofop-butyl resistance of L. chinensis. Correlation analyses revealed that the constitutive or inducible expression patterns of CYP71C4, CYP71C1, GSTF1, and GSTU6 in L. chinensis were strongly associated with the resistant phenotype. For this reason, attention should be directed towards these genes to elucidate metabolic resistance to cyhalofop-butyl in L. chinensis. The findings of this study improve the understanding of mechanisms responsible for resistance to ACCase-inhibiting herbicides in grass-weed species at the molecular level, thus aiding in the development of weed management strategies that delay the emergence of resistance to this class of pest control products.

Performance of ImproGene cfDNA blood collection tubes for mutation analysis in cancer patients.

With the widely application of liquid biopsy and the development of detection technology, the standardization of pre-analysis procedures is necessary. For controlling pre-analysis variation of circulating tumor DNA (ctDNA) in blood samples, the blood collection tubes for ctDNA preservation particularly contribute a lot. The objective of this study was to investigate whether ImproGene® Cell Free DNA Tube (ImproGene tube) can be used in sample collection, preservation and NGS based mutation detection for ctDNA. We investigated hemolysis and cell free DNA (cfDNA) concentration of blood samples stored in ImproGene tubes and detected ß-actin, LINE1 and exogenous gene level by qPCR. We compared cfDNA and RNA quantity between samples in ImproGene tube and Streck Cell-Free DNA BCT® (Streck tube). And 10 gene mutations and three fusion mutations analysis were compared by sequencing. When stored at room temperature within 7 days in ImproGene tubes, blood samples had no visible hemolysis and the cfDNA concentration, levels of ß-actin, LINE1 and exogenous gene remained stable which means no genomic DNA release and cfDNA was protected. There was no significant difference in cfDNA and RNA quantity between ImproGene tubes and Streck tubes. Furthermore, based on this limited data set, ImproGene tubes showed increased detection rates of low-level mutations. Therefore, ImproGene Cell Free DNA Tubes may have promising applications in sample collection, preservation and NGS based mutation detection for ctDNA by its good preservation performance.

Performance of ImproGene Cell-Free DNA Tubes for Stabilization and Analysis of cfDNA in Blood Samples.

BACKGROUND: With the development of liquid biopsy technology, the demand for noninvasive prenatal testing (NIPT) is increasing rapidly. The aim of the study is to evaluate the effects of different blood collection tubes on plasma cfDNA and NIPT quality control. METHODS: We investigated hemolysis, cfDNA concentration, and fragment distribution within blood samples stored in EDTA, ImproGene, and Streck tubes. The effects of ImproGene and Streck tubes on NIPT quality control were evaluated. RESULTS: The ImproGene tubes prevented the time-dependent increase of cfDNA concentration and preserved the cfDNA fragment size distribution. For NIPT quality control, there is no significant difference in cfDNA, library concentration, and fetal fraction between ImproGene and Streck tubes samples. GC content of the samples in ImproGene tubes was closer to the human genome. CONCLUSION: The ImproGene cfDNA tube has excellent performance and is an effective choice for storing blood samples for NIPT testing or other cfDNA analysis.

Systemic analysis of the expression levels and prognosis of breast cancer-related cadherins.

Cadherins form connection between cells, facilitate communication, and serve as essential agents in the progression of multiple cancers. Over 100 cadherins have been identified and they are mainly divided into four groups: classical cadherins (CDHs), protocadherins (PCDHs), desmosomal (DSC), and cadherin-related proteins. Accumulating evidence has indicated that several members of the cadherins are involved in breast cancer development. Nevertheless, the expression profiles and corresponding prognostic outcomes of these breast cancer-related cadherins are yet to be analyzed. Here, we examined the expression levels and prognostic potential of these breast cancer-related cadherins from the specific databases viz. oncomine, gene expression profiling interactive analysis, human protein atlas, UALCAN, Kaplan-Meier Plotter, and cBioPortal. We found that the CDH2/11 levels were higher in breast cancer tissues, compared to healthy breast tissues, whereas with CDH3-5, PCDH8/10, and DSC3, the levels were lower in the former than in the latter. Additionally, for CDH1/6/13/17/23, PCDH7, and FAT4, trancript level alterations between breast cancer and healthy tissues varied across different databases. The CDH1 protein levels were elevated in breast cancer tissues versus healthy breast tissues, whereas the protein levels of CDH3/11 and PCDH8/10 were reduced in breast cancer, compared to healthy breast tissues. For CDH15 and CDH23, the expression levels paralleled tumor stage. Survival analysis, using the Kaplan-Meier Plotter database, demonstrated that elevated CDH1-3 levels correlated with diminished relapse-free survival in breast cancer patients. Alternately, enhanced CDH4-6/15/17/23, PCDH10, DSC3, and FAT4 levels estimated a rise in relapse-free survival of breast cancer patients. These data suggest CDH1-3 to be a promising target for breast cancer precision therapy and CDH4-6/15/17/23, PCDH10, DSC3, and FAT4 to be novel biomarkers for breast cancer prognosis.

Layered Perovskites Enhanced Perovskite Photodiodes.

Recently, two-dimensional layered perovskites have emerged as effective additives for stabilizing conventional three-dimensional metal halide perovskites. With the addition of layered perovskites, the perovskite-based devices also exhibited enhanced optoelectronic properties, such as reduced nonradiative recombination and ionic migration, strengthened crystallinity, and anisotropic charge transport. However, the influence of the large organic cations on the performance metrics of the photodiodes is not fully understood. In this work, we systematically investigate the device performance and related optoelectronic features of the layered perovskite-enhanced perovskite photodiodes. In particular, with the addition of large organic cations to the FA0.83Cs0.17PbI3 perovskite matrix, the devices exhibited reduced dark current and noise, increased detectivity of >1013 Jones, and a consistent high speed of <100 ns. More importantly, the layered perovskite-enhanced photodiodes exhibited less hysteresis and higher breakdown voltages.

Reference genes for the study of herbicide stress responses in Leptochloa chinensis (L.) Nees and estimation of ACCase expression in cyhalofop-butyl resistant populations.

Cyhalofop-butyl resistance in Leptochloa chinensis (L.) Nees is a threat to rice production. Qualitative changes to the acetyl-CoA carboxylase gene (ACCase) have been reported to induce cyhalofop-butyl resistance in some weed species, but the role of ACCase in cyhalofop-butyl resistance through quantitative changes remains uncertain. The accurate assessment of transcriptional changes in the functional genes associated with herbicide resistance in L. chinensis is challenging owing to the lack of available reference genes for expression normalization. Here, we selected nine candidate reference genes in L. chinensis and assessed their transcription stability in populations susceptible and resistant to cyhalofop-butyl. Transcription stability was compared under conditions of herbicide stress and control conditions using BestKeeper, NormFinder, and geNorm. Elongation factor 1 alpha, eukaryotic initiation factor 4A, and cap-binding protein CBP20 were the most stable reference genes under cyhalofop-butyl treatment. Transcription levels of ACCase were evaluated in seven resistant populations, one of which showed higher transcription than the susceptible population after 24 h cyhalofop-butyl treatment. However, the slight up-regulation of ACCase (approximately 2.0-fold) is unlikely to be responsible for the high resistance levels in these populations of L. chinensis.

Assessment of Carbendazim Residues and Safety in Celery Under Different Cultivation Conditions.

Although the carbendazim is widely used to manage spot blight in celery cultivation, information on residues identified is of interest. In this study, we examined the dissipation and residual amounts of carbendazim in celery and soil under different cultivation methods when using the suggested dose and ten times of that and the bioconcentration factor of carbendazim for celery. The results showed that when celery leaves were sprayed with the suggested dose, the half-lives in a celery field and greenhouse were 2.75 days and 3.29 days, respectively. When the soil matrix was sprayed with the recommended dose before cultivation, the half-lives of carbendazim residues were 16.86 days and 11.97 days. We also conducted a long-term dietary risk assessment using the corresponding criteria. The results showed that, in China, the use of carbendazim at a dose of 0.022 g/m2 is safer and more reasonable when the harvest interval is 28 days.

Comprehensive Study of Isocarbophos to Various Terrestrial Organisms: Enantioselective Bioactivity, Acute Toxicity, and Environmental Behaviors.

The enantioselective bioactivity, toxicity, and environmental behaviors of isocarbophos (ICP) were investigated. The order of the bioactivity and toxicity was S-(+) ≥ rac > R-(-), and the difference of R-(-) and S-(+) was up to 232 times. The usage of S-(+)-ICP may efficiently reduce the usage amount of rac-ICP by 35% under the same effect, and the toxicity was not increased. Based on the toxic unit analysis, the additive effect and synergistic effect of ICP enantiomers were found in the four nontarget organisms, and R-(-)-ICP might cooperate the side-effects of S-(+)-ICP. The accumulation of rac-ICP in earthworms was enantioselective with an enantioenrichment of R-(-)-ICP, so the usage of racemic ICP might increase the exposure risk of R-(-)-ICP to earthworms. From the comprehensive results, the production of enantiomer enriched S-(+)-ICP might increase bioactivity and reduce environmental pollution, while the toxicity of S-(+)-ICP to other nontarget organisms needs to be further assessed.

A systemic study of enantioselectivity of isocarbophos in rice cultivation: Enantioselective bioactivity, toxicity, and environmental fate.

The enantioselective bioactivity and acute toxicity to target and non-target model species, and environmental fate of isocarbophos (ICP) in rice cultivation were investigated systematically. Bioactivity and toxicity of S-(+)-ICP was significantly greater than R-(-)-ICP, and the difference was 2.9-101 times. Based on the toxic unit analysis, the toxic interaction of ICP enantiomers for target pests was synergistic effect, while for non-target fish was concentration addition or antagonistic effect. Rac-ICP displayed equivalent bioactivity to S-(+)-ICP under the equal dosage, but the toxicity of rac-ICP to the tested fishes reduced at least 2 times. Rac-ICP is more suitable than optically pure S-(+)-ICP for rice cultivation based on the toxicity and bioactivity results. In environmental behavior experiments, the main metabolite of ICP, isocarbophos oxon (ICPO) was detected in rice plants, water, rice and rice hull samples. S-(+)-ICP and S-(+)-ICPO were more persistent than the R-form in these matrices. The comprehensive data of ICP enantiomers in rice cultivation will improve environmental and ecological risk assessment, and using racemate may be more safe and reasonable in rice cultivation system.

Optimal PSF Estimation for Simple Optical System Using a Wide-Band Sensor Based on PSF Measurement.

Simple optical system imaging is a method to simplify optical systems by removing aberrations using image deconvolution. The point spread function (PSF) used in deconvolution is an important factor that affects the image quality. However, it is difficult to obtain optimal PSFs. The blind estimation of PSFs relies heavily on the information in the image. Measured PSFs are often misused because real sensors are wide-band. We present an optimal PSF estimation method based on PSF measurements. Narrow-band PSF measurements at a single depth are used to calibrate the optical system; these enable the simulation of real lenses. Then, we simulate PSFs in the wavelength pass range of each color channel all over the field. The optimal PSFs are computed according to these simulated PSFs. The results indicated that the use of the optimal PSFs significantly reduces the artifacts caused by misuse of PSFs, and enhances the image quality.

Identification of Conserved and Diverse Metabolic Shift of the Stylar, Intermediate and Peduncular Segments of Cucumber Fruit during Development.

Cucumber (Cucumis sativus L.) is one of the most important vegetables and contains a high content of nutritionally beneficial metabolites. However, little is known about the metabolic variations among different parts of cucumber fruit and their kinetics during growth. In this study, the dynamic metabolic profiles in the stylar end, the intermediate segment and the peduncular end of cucumber fruit during the development were investigated by employing a non-targeted metabolomics approach, where 238 metabolites were identified. Statistical analyses revealed that both development time and tissue type influenced metabolic changes, while development time seemed to exert more effects than tissue type on the cucumber fruit metabolome. The levels of the most of the detected metabolites decreased gradually, while those of some amino acids, carbohydrates and flavonoids increased across development. The metabolomes of the stylar end and the intermediate segment were similar, although all three parts of the cucumber fruit were separated from each other in orthogonal partial least squares projection to latent structures-discriminant analysis (OPLS-DA) plots. Metabolites association analysis revealed that sn-1 and sn-2 lysophospholipids are synthesized via independent pathways in cucumber fruit. In sum, this study demonstrated both conserved and diverse metabolic kinetics of three parts of cucumber fruit, which will facilitate further study of the regulation of cucumber fruit development as well as their potential applications in nutritious quality improvement of cucumber fruit.

Orthonormal polynomials describing polarization aberration for M-fold optical systems.

Polarization aberration (PA) is a serious issue that affects imaging quality for optical systems with high numerical aperture. Numerous studies have focused on the distribution rule of PA on the pupil, but the field remains poorly studied. We previously developed an orthonormal set of polynomials to reveal the pupil and field dependences of PA in rotationally symmetric optical systems. However, factors, such as intrinsic birefringence of cubic crystalline material in deep ultraviolet optics and tolerance, break the rotational symmetry of PA. In this paper, we extend the polynomials from rotationally symmetric to M-fold to describe the PA of M-fold optical systems. Two examples are presented to verify the polynomials.

Orthogonal polynomials describing polarization aberration for rotationally symmetric optical systems.

Optical lithography has approached a regime of high numerical aperture and wide field, where the impact of polarization aberration on imaging quality turns to be serious. Most of the existing studies focused on the distribution rule of polarization aberration on the pupil, and little attention had been paid to the field. In this paper, a new orthonormal set of polynomials is established to describe the polarization aberration of rotationally symmetric optical systems. The polynomials can simultaneously reveal the distribution rules of polarization aberration on the exit pupil and the field. Two examples are given to verify the polynomials.

Simultaneous enantioselective determination of triadimefon and its metabolite triadimenol in edible vegetable oil by gel permeation chromatography and ultraperformance convergence chromatography/tandem mass spectrometry.

A novel, sensitive, and efficient enantioselective method for the determination of triadimefon and its metabolite triadimenol in edible vegetable oil, was developed by gel permeation chromatography and ultraperformance convergence chromatography/tandem triple quadrupole mass spectrometry. After the vegetable oil samples were prepared using gel permeation chromatography, the eluent was collected, evaporated, and dried with nitrogen gas. The residue was redissolved by adding methanol up to a final volume of 1 mL. The analytes of six enantiomers were analyzed on Chiralpak IA-3 column (150 × 4.6 mm) using compressed liquid CO2-mixed 14 % co-solvents, comprising methanol/acetonitrile/isopropanol = 20/20/60 (v/v/v) in the mobile phase at 30 °C, and the total separation time was less than 4 min at a flow rate of 2 mL/min. Quantification was achieved using matrix-matched standard calibration curves. The overall mean recoveries for six enantiomers from vegetable oil were 90.1-97.3 %, with relative standard deviations of 0.8-5.4 % intra-day and 2.3-5.0 % inter-day at 0.5, 5, and 50 µg/kg levels. The limits of quantification were 0.5 µg/kg for all enantiomers based on five replicate extractions at the lowest fortified level in vegetable oil. Moreover, the absolute configuration of six enantiomers had been determined based on comparisons of the vibrational circular dichroism experimental spectra with the theoretical curve obtained by density functional theory calculations. Application of the proposed method to the 40 authentic vegetable oil samples from local markets suggests its potential use in enantioselective determination of triadimefon and triadimenol enantiomers. Graphical Abstract Chemical structures and UPC(2)-MS/MS separation chromatograms of triadimefon and triadimenol.

A Case Study of Pharmaceutical Pricing in China: Setting the Price for Off-Patent Originators.

This article aims to define a value-based approach to pricing and reimbursement for off-patent originators using a multiple criteria decision analysis (MCDA) approach centered on a systematic analysis of current pricing and reimbursement policies in China. A drug price policy review was combined with a quantitative analysis of China's drug purchasing database. Policy preferences were identified through a MCDA performed by interviewing well-known academic experts and industry stakeholders. The study findings indicate that the current Chinese price policy includes cost-based pricing and the establishment of maximum retail prices and premiums for off-patent originators, whereas reference pricing may be adopted in the future. The literature review revealed significant differences in the dissolution profiles between originators and generics; therefore, dissolution profiles need to be improved. Market data analysis showed that the overall price ratio of generics and off-patent originators was around 0.54-0.59 in 2002-2011, with a 40% price difference, on average. Ten differentiating value attributes were identified and MCDA was applied to test the impact of three pricing policy scenarios. With the condition of implementing quality consistency regulations and controls, a reduction in the price gap between high-quality off-patent products (including originator and generics) seemed to be the preferred policy. Patents of many drugs will expire within the next 10 years; thus, pricing will be an issue of importance for off-patent originators and generic alternatives.